Polymerases may have suboptimal activity associated with their stability under reaction conditions. See for example, Eckert and Kunkel, “The Fidelity of DNA Polymerase and the Polymerases used in the PCR” in Polymerase Chain Reaction I: A Practical Approach, McPherson, et al., eds., Oxford University Press, New York, N.Y., (1991), pp. 235-244.
Early work reported by Capitani, et al., (Molecular and Cellular Biochemistry, 27:137-138 (1979)) described the use of phosphatidylcholine in the form of vesicles to stimulate the activity of DNA polymerase-α. U.S. Pat. No. 5,792,612 reported the use of phospholipids in a suspension or matrix in the form of unilamellar liposomes which are combined with polymerases and DNA or RNA to increase the efficiency of polymerase-dependent amplification reactions. The unilamellar liposomes were prepared by extrusion through 0.1 μM polycarbonate membranes with a LiposoFast matrix homogenizer as reported by MacDonald, et al., (Biochimica et Biophysica Acta—Biomembranes, 1061:297-303 (1991)) and were added prior to initiation of the reaction.
One of the limitations of using long-chain phosphatidylcholine phospholipids relates to their insolubility, which necessitates the use of an extruder to create pre-formed vesicles or liposomes. Another related limitation is that vesicles and liposomes have adverse effects on certain physical attributes such as light scattering in activity assays.
Polymerases for use in polymerase-dependent amplification have reportedly benefitted from the stabilizing effect of non-phospholipid detergents characterized by 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) (U.S. Pat. Nos. 6,881,559 and 7,429,468). Later-filed applications claim CHAPS under the broader description of zwitterionic detergents for use in DNA amplification (International Publication Nos. WO 2008/013885; WO 2008/077017; U.S. Publication No. 2010/0099150).
Other agents used for polymerase stabilization have also been described. U.S. Pat. No. 6,242,235 describes the use of polyethoxylated surfactants with polymerases and International Publication No. WO 2008/013885 similarly describes a polymerase with a surfactant which “has a relatively small hydrophobic head and two long hydrophilic ethylene oxide tails that do not form micelles.” The use of non-ionic polymeric detergents and anionic detergents are described in U.S. Pat. Nos. 6,127,155 and 7,846,703 and U.S. Publication Nos. 2010/0159528
There is a continuing need to refine and improve stabilizers for polymerases so as to enhance the activity of the polymerases under storage and reaction conditions.